Field of the Invention
The present invention pertains to the field of aptamer- and nucleic acid ligand (DNA and RNA ligand)-based diagnostics. More particularly, it relates to single-stranded Deoxyribonucleic acid (“DNA”) and Ribonucleic acid (“RNA”) ligand sequences, whether individual or linked together to form longer multiple binding site “receptors,” that specifically target and bind to foodborne and waterborne pathogenic bacteria or parasites such as Campylobacter jejuni, pathogenic Escherichia coli, Listeria monocytogenes, Salmonella enterica serovar Typhimurium (formerly S. typhimurium), molds or other pathogenic fungi, Cryptosporidium and Giardia parasites and related toxins produced by some bacteria (e.g., Shiga or Vero toxins) and other virulence factors (intimins, adhesions, capsules, etc.) indicating the presence of the pathogens.
These individual or linked DNA ligand (aptamer) sequences represent valuable target analyte-responsive components of diagnostic devices or biosensors. A biosensor can be defined as any device that employs a biologically-derived molecule as the sensing component and transduces a target analyte binding event into a detectable physical signal (including, but not limited to, changes in light intensity, absorbance, emission, wavelength, color, electrical conduction, electrical resistance, or other electrical properties, etc). Once bonded with the target, these DNA ligand sequences can be used to qualitatively determine the presence of target analyte, as well as to quantify the target analyte amount, in a sample using a broad variety of assay types and diagnostic or sensor platforms including, but not limited to, affinity-based lateral flow test strips, membrane blotting, surface plasmon resonance (“SPR”), magnetic bead (“MB”)-based capture, plastic-adherent sandwich assays (“PASA”), chemiluminescence (“CL”), electrochemiluminescence (“ECL”), radioisotopic, fluorescence intensity, including quantum dot (“QD”) or other fluorescent nanoparticle (“FNP”) of dye-based, fluorescence lifetime, and fluorescence polarization (“FP”) assays or enzyme-linked (ELISA-like) microplate assays. ELISA-like assays refer to microwell or microplate assays similar to traditional “Enzyme-Linked Immunosorbent Assays” or “ELISA” in which an aptamer or nucleic acid ligand is substituted for the antibody component or components, but the other components such as peroxidase or alkaline phosphatase enzymes and color-producing substrates remain the same.
In addition, these DNA ligand sequences are valuable in competitive displacement assays which are not solely dependent on high affinity (strong attractive forces between a receptor and its ligand) or high avidity (high tensile or physical strength of receptor-ligand bonds) to produce sensitive detection (sub-nanoMolar or sub-nanogram levels), because the equilibrium constant (generally Ka=106 to 108 to enable competition) must allow reasonable displacement of previously bound target materials to detect a change at or below nanogram or nanoMolar levels. In a competitive displacement assay, labeled DNA ligand plus labeled analyte complexes compete with unlabeled analyte to bind with the labeled DNA. After allowing the labeled and unlabeled analytes to come to equilibrium with the labeled DNA, the unlabeled target analyte may be quantitatively assayed by fluorescence intensity or other methods. Such assays would include competitive displacement fluorescence resonance energy transfer (“FRET”) assays or DNA ligand “beacon” FRET assays. Each of these types of assays and detection platforms has different applications in either central laboratories or as portable detectors to identify tainted foods and water either in the field (e.g. on farms or in water supplies) or in the food processing chain progressing toward the human or animal consumer.
Background Information
The DNA ligand sequences listed in Table 1 herein were derived by iterative cycles of affinity-based selection, washing, heated elution, and polymerase chain reaction (“PCR”) amplification of bound DNA ligands from a randomized library using immobilized target analytes for affinity selection and PCR amplification followed by cloning and Sanger dideoxynucleotide DNA sequencing. Sanger dideoxynucleotide sequencing refers to DNA chain termination due to lack of a 3′-OH to link incoming bases with during DNA synthesis followed by automated fluorescence reading of the DNA sequence from an electrophoresis gel containing all of the terminated DNA fragments. DNA sequencing may be accomplished by PCR doped with dideoxynucleotides lacking hydroxyl groups at the 2′ and 3′ sugar ring positions and thereby disallowing chain formation. PCR refers to the enzymatic amplification or copying of DNA molecules with a thermo-stable DNA polymerase such as Thermus aquaticus polymerase (“Taq”) with known “primer” regions or short oligonucleotides of known sequence that can hybridize to a longer target DNA sequence to enable priming of the chain reaction (exponential doubling of the DNA target copy number with each round of amplification). A randomized library can be chemically synthesized by linking together the four deoxynucleotide triphosphate bases (adenine; A, cytosine; C, guanine; G, and thymine; T) in equal amounts (25% each), so that a combinatorial oligonucleotide arises with sequence diversity equal to 4 raised to the nth power (4n) where n is the desired length of the randomized region in bases. In other words, if position 1 in an oligonucleotide is allowed to consist of A, C, G, or T (diversity=4) by equal availability of all 4 bases and these 4 possibilities are multiplied by each base linking to 4 more possible bases at position 2, then this process yields 16 possible 2-base oligonucleotides (i.e., AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT, TA, TC, TG, TT) and so on for the entire chosen length (n) of the randomized region. This combinatorial progression displays immense diversity as a function of oligonucleotide chain length. For example, an oligonucleotide decamer of 10 base length could be expected to contain 4n=410 or 1,048,576 unique DNA sequences from which to chose or select by affinity one or more sequences that bind a given immobilized target analyte. The randomized oligonucleotide or DNA is designed to be flanked on either side by short primer regions of known and fixed sequences to enable PCR amplification (exponential copying) of the rare sequences that are selected from the random library by binding to the target after the non-binding members of the random library are washed away (not selected).
Additional assays, such as ELISA-like plate assays or fluorescence (intensity and FRET) assays, may be used to screen or verify the value of particular DNA and RNA ligands or aptamer sequences for detection of a given target analyte in a given assay format or type of biosensor. Some of the sequences operate (bind and transduce the binding signal) more effectively in affinity-based (ELISA-like or fluorescence intensity) assays, while other DNA ligand sequences against the same targets function better in competitive or other assays, thereby leading to more sensitive detection with lower limits of detection (sub-nanoMolar or sub-nanogram) and less cross-reactivity or more specificity for the target analyte. Specificity means the ability to selectively exclude molecules similar in structure to the true target analyte that may interfere with the assay and give false readings. All of the listed DNA ligand nucleotide sequences have potential applications in some type of assay format, because they have survived at least 5 rounds of affinity-based selection and enrichment (by PCR amplification), although some of the sequences will undoubtedly perform better in certain assay formats or configurations (in tubes, square cuvettes, membranes, or on biochips) than others.
Combinations of the DNA ligands whether in whole or in part (i.e., their binding sites of 5-10 or more nucleotides or bases) could be linked together in a linear or 2-dimensional or 3-dimensional fashion similar to dendrimers to bind multiple epitopes or binding sites on a complex target analyte (Ag or antigen). The advantage of linking aptamers or their shorter binding pockets, loops or binding sites is that the nascent linear, 2-D or 3-D aptamer construct will likely have improved affinity or “avidity” (tensile binding strength) making it more difficult to remove or dissociate from the target antigen. The linked aptamer complex will be likely to gain specificity as well since the probability of binding to multiple epitopes with any degree of success is multiplicative. Thus, the ability to bind to epitopes A, B and C equals the product of the probability of binding to A with high affinity times the probability of binding to B with high affinity times the probability of binding to C with high affinity and that probability is clearly much less than binding to only A, B, or C or any combination of the two epitopes therein. In this way, the specificity of aptamers or DNA ligands can be increased. This approach to binding site linkage emulates that of nature in that antibodies demonstrate linkage of their “hypervariable” (HV) regions on the antigen combining sites of the immunoglobulin light and heavy chains. In the HV regions, the variability of the 20 amino acid types is quite high and essentially represents a selection of one combination from a large combinatorial library in the protein realm. The trait of HV region linkage contributes to antibody affinity, avidity and specificity. Similarly, linking aptamers or aptamer binding sites for various epitopes in one, two or three dimensions will enhance larger aptamer or DNA ligand construct affinity, avidity, and selectivity or specificity.
All of the listed DNA ligand nucleotide sequences have potential utility in some assay format, although some of the candidate sequences will perform better in certain assay formats or configurations (in tubes, cuvettes, membranes, or on biochips) than others. Assays such as ELISA-like plate assays or fluorescence (intensity and FRET) assays, may be used to verify the utility of the DNA ligand sequences. Some of the sequences function more effectively in affinity-based (ELISA-like or fluorescence intensity) assays, while other DNA ligand sequences against the same bacterial targets or analytes function better in competitive FRET assays.